Ranga Srinivas - Academia.edu (original) (raw)

Papers by Ranga Srinivas

Journal of Biological Chemistry, 1983

Journal of Virology, 1991

Friend spleen focus-forming virus (SFFV) codes for a transport-defective envelope glycoprotein de... more Friend spleen focus-forming virus (SFFV) codes for a transport-defective envelope glycoprotein designated gp52, which is responsible for the leukemogenic properties of the virus. gp52 is a monotopic integral membrane protein anchored in the membrane by a stretch of hydrophobic amino acid residues located near the carboxy terminus of the molecule. We have constructed a mutant SFFV envelope gene in which the sequences that code for the hydrophobic membrane-spanning domain have been deleted, and we expressed this gene by using recombinant vaccinia virus vectors or retroviral vectors. The mutant SFFV envelope gene was found to encode a truncated glycoprotein (gp52t) which was also transport defective; a majority of gp52t remained cell associated, while a small proportion of the molecules underwent oligosaccharide processing. The processed form of gp52t was secreted from the cells. Retroviral vectors carrying the mutant SFFV envelope gene were found to be nonpathogenic in adult mice. The...

Nature medicine, 1999

Dideoxynucleosides, which are potent inhibitors of HIV reverse transcriptase and other viral DNA ... more Dideoxynucleosides, which are potent inhibitors of HIV reverse transcriptase and other viral DNA polymerases, are a common component of highly active anti-retroviral therapy (HAART) (ref. 1). Six reverse transcriptase inhibitors have been approved for human use: azidothymidine; 2'3'-dideoxycytidine; 2'3'-dideoxyinosine; 2', 3'-didehydro-3'deoxythymidine; 2',3'-dideoxy-3'-thiacytidine; and 4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-++ +metha nol. Although drug-resistant HIV strains resulting from genetic mutation have emerged in patients treated with HAART (ref. 1), some patients show signs of drug resistance in the absence of drug-resistant viruses. In our study of alternative or additional mechanisms of resistance operating during antiviral therapy, overexpression and amplification of the MRP4 gene correlated with ATP-dependent efflux of PMEA (9-(2-phosphonylmethoxyethyl)adenine) and azidothymidine monophosphate from cell...

AIDS research and human retroviruses, 1990

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein is essential for virus entr... more The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein is essential for virus entry and the formation of multinucleated giant cells by cell fusion, one of the major virus-induced cytopathic effects. To study the effects of potential fusion inhibitors, a vaccinia virus recombinant expressing the envelope glycoprotein was generated and used to infect HeLa CD4+ cells. Syncytium induction was observed as early as 4 h postinfection and continued until the entire monolayer was fused. The N-terminus of the gp41 subunit of the HIV envelope protein is very hydrophobic, and appears to be involved in virus-induced membrane fusion. We synthesized several oligopeptide analogs of the N-terminal region of gp41 and determined their ability to inhibit HIV-induced cell fusion in CD4+ HeLa cells. A hexapeptide which was identical in amino acid sequence to the N-terminus of gp41 was found to completely inhibit cell fusion, whereas peptides with altered sequences showed reduced inhibitory...

Virology, 1983

We have investigated the pattern of glycosylation of the membrane glycoproteins encoded by a poly... more We have investigated the pattern of glycosylation of the membrane glycoproteins encoded by a polycythemic strain of spleen focus-forming virus (SFFV). These include a major species designated gp52 and its processed form which is designated gp65. The SFFV glycoproteins were found to be predominantly intracellular, although a portion of gp65 is expressed on the cell surface. gp65 was observed to be highly sialylated and resistant to digestion with endoglycosidase-H (endo-H). In contrast, gp52 was endo-H sensitive and the polyacrylamide gel electrophoresis profile of the endo-H digests suggested the presence of four glycosylation sites. Analysis of tryptic glycopeptides from gp52 by reverse-phase high-performance liquid chromatography also suggested the presence of four glycosylation sites. Glycopeptide analysis of Pronase digests of gp52 revealed two major size classes with molecular weights of 2200 and 1500, which correspond to two of the four oligosaccharide size classes reported previously for MuLV gp70&amp;#39;s (M.C. Kemp, N.G. Famulari, P.V. O&amp;#39;Donnell, and R.W. Compans, 1980, J. Virol. 34, 154). Both glycopeptide size classes were sensitive to digestion with endo-H. The glycopeptide profile of gp65 was found to be very heterogeneous and the predominant form was a 2900-dalton size class. In addition a fucosyl glycopeptide of 2500 daltons was observed in gp65, but not in F-MuLV or F-MCF glycoproteins. In the presence of the sodium ionophore monensin, the processing of gp52 to gp65 was inhibited. Instead a smaller protein of about 60,000 daltons was observed, which did not arrive at the cell surface, a situation analogous to the processing and post-translational modification reported for gp52 from anemic isolates of SFFV (S.K. Ruscetti, J.A. Field, and E.M. Scolnick, 1981, Nature (London) 294, 663).

Virus Research, 1996

We previously demonstrated that hepatitis C virus (HCV) core protein is a strong repressor of hum... more We previously demonstrated that hepatitis C virus (HCV) core protein is a strong repressor of human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) basal transcription. In this study, we have localized the HCV core protein-response domain to a region between nucleotides-65 and + 3 within the HIV-LTR. Thus, neither the upstream negative regulatory elements, or binding sites for various transcription factors (e.g. NF-~cB, USF-I, IL2/IL-2R) nor the downstream TAR regions were involved in HCV core-mediated repression. HCV core protein mediated repression of the basal transcriptional activity of HIV-1 LTR was abrogated by the Tat protein. Furthermore, HeLa-T4 cells expressing HCV core protein showed inhibition of HIV-1 replication after acute infection with cell-free HIV. A similar observation was also noted in CD4 + and CD4-lymphocytic cell lines cotransfected with an infectious molecular clone of HIV-1 and the HCV core protein expression vector. Thus, a repression of basal transcription prior to the accumulation of threshold levels of Tat protein appears to restrict HIV-1 transcription and modulate viral replication.

Immunologic Research, 1995

We have investigated antigen-independent modulation of immune responses by monoclonal antibodies ... more We have investigated antigen-independent modulation of immune responses by monoclonal antibodies directed against both viral and nonviral antigens. BALB/c mice were immunized with monoclonal IgM (i.e. Abl) specific for either Moloney murine leukemia virus-induced cell surface antigen (MCSA) or the hapten 2,4-dinitrophenyl (DNP). Injection with either Ab 1 activated a functional idiotypic (Id) network as evidenced by production of both anti-Id (Ab2) antibodies and anti-anti-Id (Ab3) antibodies. A subset of induced Ab3 (designated Abl'), exhibited specificity for antigen (virus or DNP). In mice immunized with anti-Id antibodies (Ab2), production of Ab3 and Ab I' was also observed. In the MCSA system, antibody-induced Ab 1' responses were effective in protecting mice from tumor development upon subsequent challenge with live virus. Furthermore, antigen-independent modulation of immunity to both viral and nonviral antigens was found to be thymus-dependent. Similar findings in other viral systems suggest that antibody-induced activation of Id networks may prove a viable alternative vaccine strategy that can elicit antigen-specific responses, and in some cases protection, in the apparent absence of exposure to antigen.

European Journal of Clinical Microbiology & Infectious Diseases, 2004

A critical goal of HIV vaccine development is the identification of safe and immunogenic vectors.... more A critical goal of HIV vaccine development is the identification of safe and immunogenic vectors. Recombinant vaccinia virus is a highly effective vaccine vector, with demonstrated capacity to protect animals from various viral pathogens, including rabies. Unlike many other candidate vaccine vectors, vast human experience exists with the parenteral smallpox vaccine. However, consideration of recombinant vaccinia virus as a modern vaccine is complicated by the relatively high prevalence of immunocompromised persons compared to such prevalence 4 or more decades ago (when smallpox vaccination was still routine). Administering vaccine by the subcutaneous (SQ) route, rather than the traditional scarification route, could address these concerns. SQ administration could prevent transmission of vaccinia virus to potentially vulnerable persons; it could also avoid the most common adverse events, which are cutaneous in nature. However, previous studies suggest that elicitation of immune response against passenger gene products following SQ administration requires development of a superficial pox lesion, defeating the intention of SQ administration. This is the first report to demonstrate that SQ administration of recombinant vaccinia virus does elicit immune response to the passenger protein in the absence of a cutaneous pox lesion. Results further show that a multi-envelope HIV vaccine can elicit antibody responses toward heterologous HIV-1 not represented by primary sequence in the vaccine. These findings have global implications because they support the consideration of recombinant vaccinia virus as a valuable HIV vaccine vector system.

Antimicrobial Agents and Chemotherapy, 2000

Antimicrobial Agents and Chemotherapy, 1993

Bis(pivaloyloxymethyl) [bis(pom)] derivatives of various acyclic nucleoside phosphonates--9-(2-ph... more Bis(pivaloyloxymethyl) [bis(pom)] derivatives of various acyclic nucleoside phosphonates--9-(2-phosphonylmethoxyethyl)adenine (PMEA), 9-(2-phosphonylmethoxypropyl)adenine (PMPA), and 9-(2-phosphonylmethoxypropyl)diaminopurine (PMPDAP)--were found to exhibit 9- to 23-fold greater antiviral activity than their corresponding unmodified compounds. The cytotoxicity of the bis(pom) analogs was also increased by various degrees, thus altering the therapeutic indexes of these compounds. Metabolic studies using [3H]bis(pom)PMEA and [3H]PMEA as model compounds suggested a > 100-fold increase in the cellular uptake of the bis(pom) derivative and formation of active diphosphorylated metabolite. However, the bis(pom) derivatives were chemically unstable and highly susceptible to serum-mediated hydrolysis, factors which limit their potential utility for intracellular drug delivery.

AIDS Research and Human Retroviruses, 1994

We have reported that amphipathic helical segments in the cytoplasmic domain of the HIV-1 envelop... more We have reported that amphipathic helical segments in the cytoplasmic domain of the HIV-1 envelope glycoproteins bind to calmodulin (CaM) with high affinity, and inhibit calmodulin-regulated proteins. To investigate the possible role of calmodulin activity in HIV-1 replication, we investigated the anti-HIV activity of various CaM antagonists-trifluoperazine and naphthalenesulfonamide W13 or W7-in HeLa T4 cells, PBMCs, and various T lymphocytic cell lines. The different CaM antagonists were found to inhibit the proliferation of the different cell types to varying extent. Also, the CaM antagonists were found to exert a greater antiproliferative effect on H9/HIV-lurB, as compared to uninfected H9 cells, suggesting a deficit of CaM function in HIVinfected cells. The CaM antagonists inhibited virus-induced cell fusion in HeLa T4 cells infected with a recombinant vaccinia virus expressing HIV-1 envelope proteins at threshold concentrations that do not inhibit cell proliferation. The fusion-inhibitory effects of the CaM antagonists were also observed in cocultures of HIV-infected (H9/IIIV-1 ,,,") and uninfected H9 cells. Under these conditions, the synthesis and surface expression of the viral glycoproteins were not affected, although the kinetics of processing of HIV envelope precursor was delayed. Virus production from both HIV-infected peripheral blood mononuclear cell (PBMC) and MT-2 cell cultures was inhibited by CaM antagonists at concentrations that were inhibitory to cell proliferation. Surprisingly, threshold concentrations of CaM antagonists that do not inhibit cell proliferation were found to enhance virus production from HIV-infected MT-2 cells, but not PBMCs. These results suggest that intracellular CaM may regulate the extent of virus replication and cytopathology in HIV-infected cells.

AIDS Research and Human Retroviruses, 1996

HTLV-IIIB is perhaps the most widely used laboratory stock virus in HIV-1 research. Numerous vacc... more HTLV-IIIB is perhaps the most widely used laboratory stock virus in HIV-1 research. Numerous vaccines, enzyme-linked immunosorbant assays (ELISAs), neutralization assays, and challenge stocks have derived from HTLV-IIIB. In many cases, HIV researchers target their studies toward HTLV-UIB in order that they may compare their results with those of other laboratories. In past years, the envelope sequences of four clones (BH10,

Journal of Virology, 1982

The transport of the gp70 glycoprotein to the cell surface and concomitant release of infectious ... more The transport of the gp70 glycoprotein to the cell surface and concomitant release of infectious virus was inhibited by treatment of Friend murine leukemia virus-infected Eveline cells with the sodium ionophore monensin. Virus yields were reduced more than 50-fold by 10(-5) M monensin, whereas particle production was reduced by 50% in monensin-treated cells. The resulting particles failed to incorporate newly synthesized gp70 and p15(E), whereas the other structural proteins, p30, p15, p12, and p10, were incorporated into virions. However, monensin did not inhibit the incorporation into virions of preformed gp70. A reduction in the efficiency of cleavage of the PrENV glycoprotein precursor and a defect in the processing of simple endo-H-sensitive to complex endo-H-resistant oligosaccharides suggest that intracellular transport of gp70 may be blocked before its entry into the Golgi apparatus. Fewer particles were found to bud from the cell surface, but intracellular vacuoles with bud...

Journal of Biological Chemistry, 1989

The gp52 envelope glycoprotein of Friend spleen focus-forming virus (SFFV) is a recombinant molec... more The gp52 envelope glycoprotein of Friend spleen focus-forming virus (SFFV) is a recombinant molecule derived from Friend murine leukemia virus (MuLV) by various deletions, insertions, and substitutions. The SFFV gp52 glycoprotein, unlike MuLV envelope glycoproteins, is defective in transport to the cell surface. Only 3-57' of gp52 eventually reaches the cell surface as a processed form (gp65). Although gp52 lacks cytoplasmic tail residues found in MuLV glycoproteins, we have previously shown that this deletion is not responsible for its defective transport. In order to investigate the basis for the defective transport of gp52, we have examined the folding and assembly of gp52 molecules into oligomeric molecules. CV-1 cells infected with vaccinia virus recombinants expressing SFFV gp52 were pulse labeled and the cell extracts were fractionated by velocity centrifugation through sucrose gradients. Immediately after a 10-min pulse, gp52 was detected as a monomer in the upper part of the sucrose gradient (fractions 12 and 14) and it remained as such after a 2-h chase period. However, the processed form, gp65, was found in a lower part of the gradient (fraction 8) after a 2-h chase. The position of gp65 was found to correspond to the position of trimeric influenza hemagglutinin which was analyzed on a parallel sucrose gradient, suggesting that gp65 also exists as a trimer in this fraction. These results indicate that changes in the external domain of gp52 result in improper folding of the glycoprotein molecule, and suggest that this lack of oligomerization is responsible for the defective transport of the molecules. Only those molecules that do form oligomeric structures are transported to the Golgi complex and undergo further oligosaccharide processing, and transport to the cell surface. Friend spleen focus-forming virus (SFFV)' is a defective murine leukemia virus (MuLV) that causes an acute erythroleukemia in newborn and adult mice (Friend, 1957; Rauscher, 1962; Mirand et al., 1968; Steeves et al., 1971). SFFV has been identified as a recombinant virus containing substitutions in the envelope gene as well as deletions in the enu, gag, and pol regions of the genome (Clark and Mak,

Journal of Virology, 1987

Friend murine spleen focus-forming virus (SFFV) encodes a glycoprotein designated gp52, which is ... more Friend murine spleen focus-forming virus (SFFV) encodes a glycoprotein designated gp52, which is responsible for the leukemogenic properties of the virus. gp52 lacks a cytoplasmic domain and is defective in its transport to the cell surface. We constructed a chimeric envelope gene which codes for a molecule with an external domain derived from the SFFV envelope gene and membrane-spanning and cytoplasmic domains derived from the Friend murine leukemia virus envelope gene. Like gp52, the chimeric protein was defective in its transport to the cell surface, indicating that the absence of a cytoplasmic tail is not responsible for the defective intracellular transport of SFFV gp52. However, unlike wild-type SFFV, the chimeric SFFV genome failed to induce erythroleukemia in adult mice. The results indicate that the altered membrane-spanning domain, lack of a detectable cytoplasmic tail in gp52, or both factors are prerequisites for the erythroleukemia-inducing properties of SFFV but are no...

Antimicrobial Agents and Chemotherapy, 1998

9-R-2-Phosphonomethoxypropyl adenine (PMPA) is an acyclic nucleoside phosphonate analog that has ... more 9-R-2-Phosphonomethoxypropyl adenine (PMPA) is an acyclic nucleoside phosphonate analog that has demonstrated efficacy against human immunodeficiency virus (HIV). We recently described the synthesis, metabolism, and biological activities of bis(isopropyloxymethylcarbonyl)PMPA [bis(poc)PMPA] as an orally bioavailable prodrug for PMPA. Among a large panel of drug-resistant HIV type 1 variants, only the K65R virus was resistant to PMPA. K65R virus also showed reduced susceptibility to bis(poc)PMPA, although the prodrug could still inhibit these viruses at submicromolar, nontoxic concentrations. Among a panel of seven primary clinical isolates from patients with diverse treatment histories, only one isolate showed reduced susceptibility to PMPA and was found to carry three mutations (M41L, T69N, R73K) in its reverse transcriptase catalytic domain.

Current Topics in Microbiology and Immunology, 1991

Encyclopedia of Virology, 1999

Virology, 1988

Friend spleen focus-forming virus (F-SFFV) encodes a glycoprotein designated gp52, which is defec... more Friend spleen focus-forming virus (F-SFFV) encodes a glycoprotein designated gp52, which is defective in its intracellular transport and accumulates in the rough endoplasmic reticulum. Only 3-5% of the mature form of gp52 eventually reaches the cell surface. Compared to transport-competent murine leukemia virus (MuLV) glycoproteins, the gp52 molecule exhibits several structural differences which may have resulted in the possible loss of signals required for transport to the cell surface. To determine the effect of these alterations on the specific sites of surface expression of the molecule, the SFFV env gene was expressed from a vaccinia virus recombinant in a polarized epithelial cell line in which retrovirus glycoproteins are expressed exclusively on basolateral surfaces. We also determined the site of expression of a chimeric env protein which contains the external domain of SFFV gp52 the transmembrane, and the cytoplasmic tail residues of Friend MuLV. The wild-type and chimeric env gene products were defective in transport, and remained primarily in an unprocessed form in MDCK cells or CV-1 cells. However, both glycoproteins were detected at low levels on the basolateral surfaces of MDCK cells, a line of polarized epithelial cells. These results indicate that the presence or absence of a cytoplasmic tail as well as a 585-base deletion in the external domain has no affect on the site of polarized expression of a murine retrovirus glycoprotein.